EP2167526A1 - Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal - Google Patents

Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal

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Publication number
EP2167526A1
EP2167526A1 EP08786090A EP08786090A EP2167526A1 EP 2167526 A1 EP2167526 A1 EP 2167526A1 EP 08786090 A EP08786090 A EP 08786090A EP 08786090 A EP08786090 A EP 08786090A EP 2167526 A1 EP2167526 A1 EP 2167526A1
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EP
European Patent Office
Prior art keywords
factor viii
protein
salt
purification
ethylene glycol
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EP08786090A
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German (de)
English (en)
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EP2167526B1 (fr
Inventor
Susanne Bang
Lars Thim
Johan Karlsson
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Novo Nordisk AS
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Novo Nordisk AS
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Priority to EP08786090A priority Critical patent/EP2167526B1/fr
Priority to PL08786090T priority patent/PL2167526T3/pl
Publication of EP2167526A1 publication Critical patent/EP2167526A1/fr
Application granted granted Critical
Publication of EP2167526B1 publication Critical patent/EP2167526B1/fr
Priority to NL300663C priority patent/NL300663I2/nl
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Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745Blood coagulation or fibrinolysis factors
    • C07K14/755Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Definitions

  • the present invention is related to a method for purifying a recombinant protein using a multimodal or mixed mode resin containing ligands which comprise a hydrophobic part and a negatively charged part.
  • the protein of interest is a coagulation factor, particularly relevant to the purification of compositions of recombinant Factor VIII.
  • Human Factor VIII also known as antihaemophilia factor or FVIII:C is a human plasma protein consisting of two polypeptides with light chain molecular weight of 80,000 daltons and a heavy chain molecular weight variable from 90,000 to 220,000. It is considered as one of the key cofactors in the coagulation pathway necessary for the conversion of Factor X into its active form Factor Xa.
  • Factor VIII circulates in plasma as a non-covalent complex with von Willebrand Factor (also known as FVIII : RP).
  • Hemophilia a bleeding disorder is caused due to abnormal levels of Factor VIII. Factor VIII levels below 20% normal may result in hemophilic condition in humans. A drop in the levels of less than 1% of Factor VIII leads to severe bleeding disorder, with spontaneous joint bleeding being the most common symptom.
  • isolation and purification of Factor VIII has been from a plasma derived source (cryoprecipitate).
  • Purification procedures from plasma-derived sources include those exploring the use of immunoaffinity purification using polyclonal and monoclonal antibodies for the purification of FVIII.
  • the Factor VIII effluent contains some residual antibody due to leaching from the support matrix, which may result in antigenicity during ultimate use, i.e when introduced into human or animal system.
  • Purification procedures exploring the use of ion exchange chromatography on e.g. agarose beads have also been used for purification of factor VIII from plasma. These methods, however, often suffer from certain levels of contamination of the resulting FVIII: C.
  • the contaminants in recombinantly produced protein may include secreted proteins in the culture medium, media components, cell lysates, unwanted proteins produced by the cells and the nucleic acids.
  • the aqueous source materials in which the polypeptides of interest are found are furthermore often seen to be contaminated with one or more viruses.
  • Techniques for inactivating viruses in polypeptide mixtures are known in the art, such as e.g. chemical methods, using solvent/detergent solutions, irradiation methods, or thermal methods, but attempts to combine such techniques with known polypeptide purification processes have produced methods with a multiplicity of steps unsuitable for large-volume production. It is also important to exert caution in that the used viral-inactivating agents do not denature the protein or are difficult to separate from the protein of interest.
  • Nordfang et al. Thrombosis and Haemostasis 58(4), 1043-1048 (1987) describes a separation using an antibody resin and a buffer containing 50% ethylene glycol and high salt.
  • the purification of a recombinant protein expressed in mammalian cell system is typically performed in several steps.
  • the different steps are usually separated into capture, intermediate and polishing.
  • the objective of the capture step is two-fold; a) to obtain the target protein in a stable solution form and b) to reduce the volume (i.e. obtaining a solution concentrated with respect to protein content compared to the solution loaded onto the column ("the loading").
  • the latter step (reduction of volume) is critical to facilitate the subsequent steps of purification.
  • the capture step is commonly achieved by using chromatography with an ion- exchange resin.
  • the drawback of using an ion-exchange resin is that the conductivity and/or pH of the loading has to be adjusted. When the conductivity is adjusted, in most instances reduced, it is performed by addition of water which increases the volume of the starting material, making it impractical for subsequent steps and overall cumbersome for production purposes. Furthermore, adjustment of pH often results in the formation of aggregates which could interfere with the performance of the purification steps.
  • the polishing step refers to a final purification step, wherein trace contaminants and impurities are removed and the yield is an active biological product. Contaminants removed during the polishing step are often conformers of the target molecule or suspected leakage products.
  • the method of the present invention is advantageous in that it, in a single step, provides a volume reduction and a considerable increase in specific activity.
  • the method in a single step, combines a capture and a purification step.
  • the present invention also provides an efficient process for producing a highly concentrated and very pure solution of recombinant Factor VIII wherein the Factor VIII protein is stabilized against degradation.
  • the instant invention is related to the novel and efficient purification of recombinant proteins, especially coagulation factor VIII.
  • the inventors of the present invention have found that use of multimodal columns for the capture of factor VIII molecules from mammalian cell culture fluid is a surprisingly fast and efficient purification method for removing contaminants from FVIII protein harvested from the culture, without the loss in the activity of the protein.
  • the invention provides a method of purifying a coagulation Factor VIII protein containing one or more contaminants, the method comprising the steps of:
  • the method further comprises a step (c) wherein the Factor VIII- containing solution resulting from step (b) is collected; and/or the method further comprising a step (al) wherein the column, subsequent to step (a) and before step (b), is passed with one or more wash buffer(s).
  • one or more of said wash buffer(s) comprise(s) 1OmM to 100OmM salt
  • the elution buffer of step (b) contains at least 2M salt and at least 45% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof, such as, e.g. 2.3-2.6 M salt and 48-52% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof
  • the salt contained in the elution buffer of step (b) is selected from: NaCI, NH 4 CI, KCI, (NH 4 ) 2 SO 4 , CH 3 CO 2 NH 4, or a mixture of two or more of these.
  • the invention provides a single step method of purifying a Factor VIII protein containing one or more contaminants, comprising the steps of: a) contacting the protein with a multi-modal resin containing the ligands which comprise a hydrophobic part and a negatively charged part; b) eluting the said protein with an elution buffer containing at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof, and calcium ions; wherein the said method achieves a reduction in the column volume of about 250-fold.
  • the invention provides a single step method of purifying a Factor VIII protein containing one or more contaminants, comprising the steps of: a) contacting the protein with a multi-modal resin containing the ligands which comprise a hydrophobic part and a negatively charged part; b) eluting the said protein with an elution buffer containing at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof, and calcium ions; wherein the said method achieves a "purification factor" of at least 30-fold.
  • the invention provides a method for stabilizing a Factor VIII protein, comprising the steps of: a) contacting the protein with a multi-modal resin containing the ligands which comprise a hydrophobic part and a negatively charged part; b) eluting the said protein with an elution buffer containing at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof, and calcium ions.
  • Figure IA is a chromatogram showing the UV absorption at 280nm, conductivity and pH over a purification run with equilibration, loading, wash and elution.
  • Figure IB shows an enlargement of the wash and elution part of the chromatogram of Figure 1.
  • Figure 2 shows a silver-stained SDS gel electrophoresis illustrating the purification obtained using the Capto MMC column in the purification steps of loading, flow through (FT) and elution.
  • Lanel MW standard
  • Lane 2 Cell culture medium
  • Lane 3 Empty
  • Lane 4 Break through (not bound material) from the Capto MMC column
  • Lane 5 Empty
  • Lane 6 Capture pool (protein material bound and eluted from the Capto MMC column
  • Figure 3 is a histogram showing the percentage yields of recombinant Factor VIII purified using Capto MMC resin with different elution buffers containing various concentrations of NaCI or NH 4 CI and ethylene glycol.
  • Figure 4 shows the effect of repeated freeze/thaw cycles on the stability of the Factor VIII in the capture pool.
  • the present method of purifying coagulation factor VIII is performed by using multimodal chromatography making use of resins that are both hydrophobic and negatively charged.
  • the solution of the protein to be purified e.g. the protein-containing supernatant obtained from mammalian cell fermentation, is applied to the multimodal resin by passing a solution over a chromatography column containing said resin.
  • the solution (loading) is typically filtered (e.g. by dead-end filtration or cross flow filtration) and/or centrifuged to remove cells and particles before being loaded onto the resin.
  • the loading step does not require adjustment of pH or conductivity.
  • the column is typically washed with one or more wash buffers, one of which containing high concentration of salt. In one embodiment, the column is washed, after loading, with equilibration buffer, optionally followed by wash with a second buffer with high concentration of salt.
  • elution is performed by passing an elution buffer comprising a high concentration of salt and an organic solvent over the column.
  • volume reduction is meant a reduction in the volume of protein-containing eluate solution when compared to the volume of protein-containing loading solution (e.g. the clarified crude sample from the fermentation process).
  • the purification method of the invention further provides a protein yield of 70-100% and the purification factor (increase in the specific activity) is up to 100-fold.
  • the resultant purified protein is stable in the elution buffer for at least 2 months at -70 0 C.
  • the method of the invention provides a stable factor VIII solution :
  • the capture eluate can be frozen and thawed at least 3 times without significantly loss of activity.
  • the invention makes use of multi-modal chromatography resins for the separation of the factor VIII protein of interest from the contaminants.
  • the multi-modal chromatography wherein two or more different, but co-operative, sites interact with a target, has been determined to be an appropriate system for purification of factor VIII protein.
  • the chromatography resin used for the purification of the recombinant protein in the present invention is commercially available, for example: Capto MMCTM (GE Healthcare) or MBI HyperCelTM (Pall, formerly BioSepra).
  • Capto MMC contains a ligand with multimodal functionality that gives a different selectivity compared to traditional ion exchangers and also provides the possibility of binding proteins at salt concentrations as high as 0.5M to 1.0 M.
  • the ligand herein exhibits diverse functionalities for interaction with the target molecule and thus offers ionic interaction, hydrogen bonding and hydrophobic interaction.
  • the multimodal resin is Capto MMCTM.
  • MBI HyperCelTM (Pall), yet another multimodal chromatography, is efficient in tackling stability problems and leakage from protein-based affinity columns. More specifically, MBI Hypercel R TM (Pall), an adsorbent comprising mercapto-benzimidazole-sulphonic acid ligands, provides hydrophobic as well as ionic interactions with a given protein. The hydrophobic interactions are assumed to be due to the aromatic ring system, while the ionic interactions should be due to the SO 3 - substituent, which is known as a strong cation exchanger. In addition, the nitrogen atoms of the aromatic system of the MBI ligand are chargeable under certain conditions, and can consequently provide ionic interactions with negatively charged groups,
  • the contaminants are adsorbed to the multimodal ligands, and an essentially pure fraction of coagulation factor VIII is recovered by a subsequent selective elution.
  • the capture step provides a purification factor (increase in specific activity) of at least 80 times, such as 80 to 100-fold, 90 to 100-fold or 100-160-fold
  • the resulting capture pool comprises the factor VIII protein that is at least 10% pure, such as at least 20% or about 30% to 60%, such as 30% to 50%.
  • the purity is defined at the amount of factor VIII protein in % (w/w) (e.g. as determined by ELISA) of the total amount of protein.
  • the solution loaded to the multi-modal chromatography resin is a factor Vlll-containing harvest from mammalian cell fermentation.
  • said harvest is filtered and/or centrifuged to remove cells and particles; in another embodiment the harvest is loaded to the resin without any adjustment of pH or conductivity of the loading solution.
  • the present invention specifically encompasses a method wherein the loading step comprises loading the protein-containing loading solution onto the resin at a pH and conductivity similar to the pH and conductivity a factor Vlll-containing harvest from mammalian cell fermentation, optionally a filtered harvest.
  • the Factor Vlll-containing harvest is stabilized by the addition of imidazol (such as, e.g., 1-10 mmol imidazol per L harvest). Due to the small amount and volume of added imidazol compared to the volume of the harvest, any change in pH and/or conductivity of the harvest is negligible.
  • the harvest is then (i) filtered and/or centrifuged to remove cells and particles and (ii) loaded to the resin without further addition of chemicals.
  • the method of the invention is typically performed in a number of steps: equilibration of column, loading and elution.
  • One or more washing steps may be included after loading and before elution.
  • the washing step(s) is/are typically done using at least one buffer containing a high concentration of salt.
  • a wash step is employed using a buffer comprising 10 imM to 1000 imM salt, such as, e.g. 100-1000 imM, or 250-1000 imM, or 500- 1000 mM, or 500-800 mM salt.
  • the method includes a washing step wherein, after loading, the column is first washed with equilibration buffer and then washed with a second washing buffer containing high salt.
  • the equilibration buffer comprises a buffering agent (such as e.g. imidazole in the range of 5 mM to 100 mM, e.g. 2OmM Imidazole), a Ca-salt (1 mM to 100 mM, e.g. 10 mM CaCI 2 ,) a detergent (such as e.g. TweenTM 80 in the range of 0.001% to 0.1%, e.g. 0.02% (w/v) TweenTM 80) and a salt (10 mM to 1000 mM, e.g. 50 mM NaCI).
  • a buffering agent such as e.g. imidazole in the range of 5 mM to 100 mM, e.g. 2OmM Imidazole
  • Ca-salt 1 mM to 100 mM, e.g. 10 mM CaCI 2
  • a detergent such as e.g. TweenTM 80 in the range of 0.001% to
  • the equilibration buffer is maintained at a pH of 6.0 to 8.0; preferably the pH conditions are maintained at 7.3 to 7.5. It will be appreciated by a person skilled in the art that the factor VIII protein exemplified herein is stable at a pH of about 6.0 to 8.0 and thus the equilibration buffer is at a pH of 7.3 - 7.5, more preferably at 7.5.
  • the wash buffer comprises a buffering agent (such as e.g. imidazole in the range of 5 mM to 100 mM, e.g. 2OmM Imidazole), a Ca-salt (1 mM to 100 mM, e.g.
  • a detergent such as e.g. TweenTM 80 in the range of 0.001% to 0.1%, e.g. 0.02% (w/v) Tween 80
  • a salt e.g. NaCI in the range of 50 mM to 1000 mM
  • the elution step is performed with an elution buffer comprising at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol or a mixture thereof, and calcium ions (1 mM to 100 mM, e.g. 10 mM CaCI 2) .
  • the elution buffer has a pH of 6.0 to 8.0; preferably the pH conditions are maintained at 7.3 to 7.5. To maintain the desired pH conditions, the elution buffer also contains a buffering agent.
  • buffering agent encompasses those agents which are able to maintain the pH of a solution within a desired range - in the context of the present invention within the range of 6.0 to 8.0, such as 7.3 to 7.5.
  • the buffer concentration range is chosen to maintain the preferred pH of the solution.
  • the buffering agent may also be a mixture of at least two buffering agents, wherein the mixture is able to provide a pH value in the specified range.
  • Buffering agents may include, but are not limited to, citrate (sodium or potassium), acetate (ammonium, sodium or calcium), histidine (L-histidine), phosphate (sodium or potassium), tartaric acid, succinic acid, MES, HEPES, imidazol, TRIS, ethanolamine, and mixtures of two or more thereof.
  • the buffering agent employed is imidazole in the range of 5 mM to 10O mM.
  • Preferred salts are NaCI, NH 4 CI, KCI, (NH 4 ) 2 SO 4 , CH 3 CO 2 NH 4 (ammonium acetate, NH 4 Ac), or a mixture of two or more thereof.
  • the elution buffer comprises about 2.5 M salt and at least 40% glycol; in another embodiment, the elution buffer comprises about 2.5M salt and about 50% glycol.
  • the salt is NaCI; in another, the salt is NH 4 CI.
  • the glycol is ethylene glycol; in another, the glycol is propylene glycol.
  • the elution buffer comprises NaCI and ethylene glycol.
  • NH 4 CI at a concentration of 2.5M with ethylene glycol is also used for elution of the protein.
  • the elution buffer further comprises a detergent.
  • the eluent is 2OmM Imidazole, 1OmM CaCI 2 , 0.02% (v/v) Tween 80, 2.5M NaCI and 8 M ethylene glycol at a pH of 7.5.
  • MBI HyperCelTM resin is used in the method for purification of Factor VIII.
  • binding of the recombinant factor VIII using the MBI HyperCelTM column is performed after addition of 10OmM Na 2 SO 4 to the harvest and elution is achieved with IM NaCI and 5M glycerol.
  • the method comprises the steps of: a) contacting the Factor VIII with a multimodal or mixed mode resin containing the ligands which comprise a hydrophobic part and a negatively charged part; b) passing the column with equilibrating buffer comprising 2OmM Imidazole, 10 mM CaCI 2 , 0.02% (w/v) Tween 80 and 50 mM NaCI and wash buffer comprising of 2OmM Imidazole, 10 mM CaCI 2 , 0.02% (w/v) Tween 80 and 1.5M NaCI; c) eluting the said protein with elution buffer comprising 2OmM Imidazole, 10 mM CaCI 2 , 0.02% (v/v) Tween 80, 2.5M NaCI, and 8M ethylene glycol.
  • the first step of the purification of a recombinant protein expressed in mammalian cells handles large loading volumes.
  • it is important to reduce the volume to where it can be easily handled.
  • the purification step has to have a significant yield to achieve process economy for commercial or other purposes.
  • About 20 to 100 times reduction in volume in the first step is normally aimed at in the first step of the protein purification wherein the latter is regarded as a sound volume reduction.
  • the loading volume containing the protein to be purified is above 500 column volumes (CV).
  • the protein eluted provides a reduction in the final volume of more than 100-fold.
  • the instant invention encompasses a loading volume of 50-600 CV.
  • the reduction in volume is preferably about 100-300 times.
  • the loading volume is about 500 CV, or about 450-600 CV, and the factor VIII protein is eluted in about 2CV achieving a volume reduction of about 250 times.
  • the capture step can be combined with one or more virus inactivating agents, such as e.g. Triton X-100 (for example in a concentration of about 1% (w/v).
  • virus inactivating agent may e.g. be added to one or more of the used buffers.
  • Flow rates are manipulated to be between 300-950cm/hr for all steps of the purification process except elution and clean-in-place (CIP) steps where flow rates are at 30-350cm/h.
  • the flow rate at loading is 450cm/h; during elution and CIP, the flow rate is decreased to 30cm/h. In one embodiment of the invention, the flow rate at loading is about 450-600cm/h.
  • the invention also relates to a preparation comprising :
  • a Factor VIII protein (i) a Factor VIII protein; and (ii) a buffer containing at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol, or a mixture thereof, and calcium ions; wherein the Factor VIII is 30% to 60% pure.
  • Fractor VIII protein is meant to include full-length factor VIII, FVIII: C, and deletion derivatives of full-length factor VIII having coagulant activity.
  • deletion derivative it is meant factor VIII wherein the whole or part of the B-domain is missing while the coagulant activity is retained.
  • B-domain deleted forms of Factor VIII are, for example, described in WO91/09122. The structure and biochemistry of Factor VIII in general have been described by Kaufman (Trends in Biotechnology, 9, p.353-359 (1991) and Hematology, 63, p.155-165 (1991)).
  • factor VIII sequence variants and derivatives including pegylated, acylated, and polysialylated forms of factor VIII, having maintained the characteristic factor VIII biological activity.
  • the factor VIII may be obtained by recombinant means.
  • the factor VII is recombinantly produced factor VIII.
  • the Factor VIII is full length Factor VIII; in another embodiment, the Factor VIII protein is a B-domain deleted Factor VIII.
  • the factor VIII is a pegylated, acylated, or polysialylated Factor VIII.
  • Factor VIII concentrates derived from human plasma contain several fragmented fully active Factor VIII forms as described by Andersson et al (Proc. Natl. Acad. Sci.
  • the smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by metal ion(s) (see, e.g. EP197901).
  • metal ion(s) see, e.g. EP197901.
  • Such Factor VIII fragments are also included - purification of such fragments constitute one embodiment of the present invention.
  • Biological activity of factor VIII are assayed, e.g., as described in Experimental section (see below).
  • the term "eluent” is meant in its conventional meaning as it is identified in the art which is a buffer or buffers of suitable pH and/or ionic strength to release one or more proteins or compounds from a separation matrix.
  • the "salt" in the elution step refers to an alkaline earth, alkali metal, or ammonium salt, i.e., a salt having a cation from the alkaline earth or alkali metal elements or an ammonium cation and having an inorganic or organic (hydrocarbon-based) anion.
  • salts include sodium chloride, ammonium chloride, sodium citrate, potassium citrate, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, calcium phosphate, ammonium phosphate, magnesium phosphate, potassium phosphate, sodium sulfate, ammonium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, etc.
  • Preferred salts herein are chlorides or sulfates. The most preferred salt herein is sodium chloride.
  • multi-modal chromatography ligand refers to a ligand that is capable of providing at least two different, but co-operative, sites which interact with the protein to be bound. One of these sites gives an attractive type of charge-charge interaction between the ligand and the substance of interest. The other site typically gives electron acceptor-donor interaction and/or hydrophobic and/or hydrophilic interactions.
  • the present invention is directed, particularly to the hydrophobic interactions. Electron donor-acceptor interactions include interactions such as hydrogen-bonding, .pi. -.pi., cation-. pi., charge transfer, dipole- dipole, induced dipole etc.
  • Multi-modal chromatography ligands as used herein or elsewhere are also known as "mixed mode" chromatography ligands.
  • capture step refers, in the context of liquid chromatography, to the initial step of a separation procedure. Most commonly, a capture step includes clarification, concentration, stabilization and a significant purification from soluble contaminants. After the capture step, an intermediate purification may follow, which removes most of the significant impurities including DNA, viruses and endotoxins. These impurities can also be remove/reduced in capture.
  • polishing step refers to a final purification step, wherein trace contaminants and impurities are removed and the yield is an active biological product. Contaminants removed during the polishing step are often conformers of the target molecule or suspected leakage products.
  • Column Volume refers to the geometric volume of the part of the tube that contains the packing. As per the instant invention, the loading volume is above 50 Column Volumes (CV) or more or less about 300-600 CV or 450- 600 CV.
  • the protein eluted at the final step of purification is about 2-5 CV achieving a volume reduction of about 100-fold to 350-fold, such as, e.g., 250-fold.
  • the "purification factor” is defined as increase in specific activity: That is U/mg protein before the purification step compared to U/mg protein after the purification step.
  • the specific activity of coagulation factor VIII may be assayed by commercially available activity assays, such as, for example, using CoaTest ® ChromogenixTM (Instrumentation Laboratory, Belgium) according to the manufacturer's protocol or as described in the Experimentals section, below.
  • a single step method refers to a method of purifying a Factor VIII protein containing one or more contaminants, wherein the method achieves both a reduction in volume and an increase in specific activity (purification factor > 1).
  • the method provides (i) a reduction in the column volume of about 250-fold, and (ii) a "purification factor" of at least 30-fold.
  • factor VIII isolation of factor VIII as well as recombinant production of factor VIII (full- length and variants/truncated forms) are described in the art:
  • Recombinant DNA technology has allowed construction of plasmids that direct the expression of fusion products of Factor VIII protein in transfected mammalian cells.
  • B domain is missing and/or specific amino acid positions are modified, for example, such that normally protease-labile sites are resistant to proteolysis, e.g. by thrombin or activated Protein C.
  • Other analogs include modification at one or more lysine and/or tyrosine residues.
  • the truncated form of the protein is a 170 kDa glycoprotein consisting of 1438 amino acids.
  • This B-domain deleted protein is a post- translational modification of the plasma-derived Factor VIII and is comparable in its antihemophilic activity to that of the parent.
  • the activity of purified Factor VIII may e.g. be assayed using CoaTest ® ChromogenixTM (Instrumentation Laboratory, Belgium) according to the manufacturer's protocol.
  • the recombinant Factor VIII was expressed in CHO-cells in a commercially available serum free media.
  • the protein harvest from the fermentation was further used for the purification.
  • Capto MMC (GE Healthcare, Uppsala, Sweden) resin was used for the purification of the recombinant protein.
  • the column was packed with a bed height of 12cm and 24ml bed volume.
  • the purification process involved loading wherein the harvest from the fermentation was applied onto the column.
  • the column volume (CV) of loading was 450CV (Table 1). This was followed by passing the column with equilibration buffer consisting of 2OmM imidazole, 1OmM CaCI2, 0.02% (v/v) Tween 80 and 5OmM NaCI. The pH of the equilibration buffer is adjusted to 7.5. The column is further washed with wash buffer (2OmM imidazole, 1OmM CaCI2, 0.02% (v/v) Tween 80 and 1.5M NaCI) at a pH equivalent to the equilibration buffer, i.e. 7.5. The column volumes of equilibration and washing steps were 3CV (Table 1).
  • Elution buffer consists of
  • Flow rate was maintained at 450cm/h at every step of purification except the elution and CIP during which the flow rate was 30cm/h.
  • the activity of purified Factor VIII was measured using a modification of the CoaTest ® SP assay:
  • Reagents and buffer stock solution from the CoaTest ® SP kit were used. All reagents were allowed to reach room temperature before initiation of experiment. Samples were diluted in CoaTest assay buffer (50 imM Tris, 150 imM NaCI, 1 % BSA, pH 7.3, with preservative) to approximately 2 mU/ml. The reference plasma was diluted in assay buffer to 5 - 0.5 mU/ml. The samples were diluted in assay buffer to 12 and 9 U/ml, then 10-fold in FVIII-deficient plasma with VWF (Dade Behring), and finally 10- and 20-fold in CoaTest ® assay buffer (resulting in 4 dilutions per sample).
  • CoaTest assay buffer 50 imM Tris, 150 imM NaCI, 1 % BSA, pH 7.3, with preservative
  • the reference plasma was diluted in assay buffer to 5 - 0.5 mU/ml.
  • the samples were diluted in assay buffer to 12
  • the absorbance at 415 nm was measured on a Spectramax ® microtiter plate reader (Molecular Devices) with absorbance at 620 nm used as reference wavelength.
  • the value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration in mU/ml.
  • Factor VIII antigen was determined using the commercial ELISA kit from Affinity Biologicals (VisuLize Factor VIII Antigen Kit, Lot AG8-0006) essentially as described by the manufacturer.
  • Recombinant Factor VIII was purified from the fermentation harvest as described in Example 2, above. Purification factor and yield of Factor VIII protein was determined by CoA and ELISA as described above:
  • SDS gel electrophoresis was carried out using 7% NuPage ® tris acetate gel with tris acetate as running buffer essentially as described by the manufacturer (Invitrogen). Briefly, the gel electrophoresis was carried out for 50 min at 150 volt and the samples were stained with SilverQuest ® staining kit (Invitrogen) as described in the manufacturer's instructions. The molecular weight (MW) standards used were Mark 12 from Invitrogen.
  • Protein samples for electrophoresis were denatured and reduced at 70 0 C for 10 min in LDS sample buffer (Invitrogen) containing 50 mM DTT. Separation gels were 7% tris-acetate (TA) Pre-Cast Novex polyacrylamide gel (Invitrogen) and electrophoresis was carried out for 60 min at a limiting voltage of 150 V and with tris-acetate buffer (Invitrogen) as both anode and cathode buffer. Silver staining was carried out using SilverQuest ® (Invitrogen) according to the manufacturer's description. The gel illustrate the large purification obtained using the Capture MMC column. The cell medium contains a series of proteins. The large majority of these protein pass through the column whereas the Factor VIII is highly concentrated in the capture pool from the column.
  • Elution buffer of 2OmM ethanolamine, 1OmM CaCI 2 , Tween 80 (0.2%v/v), at pH 8.3 with different salt and ethylene glycol concentrations were used which include: a. no addition; b. IM NH 4 CI; c. 2.5M NH 4 CI; d. 3M ethylene glycol; e. IM NH 4 CI + 3M ethylene glycol; f. 2.5M NH 4 CI + 3M ethylene glycol; g. 8M ethylene glycol; h. IM NH 4 CI + 8M ethylene glycol; I. 2.5M NH 4 CI + 8M ethylene glycol; 2.5M NaCI + 8M ethylene glycol.
  • the elution conditions were determined based on the percentage yield.
  • Figure 3 shows that at concentrations of 2.5M NH 4 CI or 2.5M NaCI and 8M ethylene glycol, the percentage yield of the protein was maximum (Figure 3).

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Abstract

L'invention porte sur un procédé de purification d'une protéine recombinante à l'aide d'une résine multimodale ou en mode mixte contenant des ligands qui comprennent une partie hydrophobe et une partie chargée négativement. L'invention est avantageuse par le fait que c'est un procédé chromatographique à une seule étape qui ne nécessite pas d'ajustement de pH ou de conductivité pendant l'étape de charge et conduit à rendement élevé et une puissance élevée. Le procédé est utilisé pour la purification de compositions recombinantes de facteur de coagulation, en particulier le Facteur VIII recombinant.
EP08786090A 2007-07-11 2008-07-11 Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal Active EP2167526B1 (fr)

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EP08786090A EP2167526B1 (fr) 2007-07-11 2008-07-11 Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal
PL08786090T PL2167526T3 (pl) 2007-07-11 2008-07-11 Oczyszczanie czynnika VIII za pomocą żywicy o mieszanej funkcji lub wielofunkcyjnej
NL300663C NL300663I2 (fr) 2007-07-11 2014-04-15

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EP07112287 2007-07-11
EP08786090A EP2167526B1 (fr) 2007-07-11 2008-07-11 Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal
PCT/EP2008/059094 WO2009007451A1 (fr) 2007-07-11 2008-07-11 Purification du facteur viii à l'aide d'une résine en mode mixte ou multimodal

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11203747B2 (en) 2012-10-24 2021-12-21 Genzyme Corporation Elution of biomolecules from multi-modal resins using MES and MOPS as mobile phase modifiers

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5619738B2 (ja) * 2008-06-24 2014-11-05 オクタファルマ アクチェンゲゼルシャフト 凝固第viii因子の精製方法
MX2011001624A (es) 2008-08-21 2011-03-28 Octapharma Ag Factor viii y ix humano producido en forma recombinante.
US8945895B2 (en) 2009-07-31 2015-02-03 Baxter International Inc. Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof
US8277649B2 (en) 2009-12-14 2012-10-02 General Electric Company Membranes and associated methods for purification of antibodies
US8974780B2 (en) 2010-02-24 2015-03-10 Zymenex A/S Process for production and purification of recombinant lysosomal alpha-mannosidase
RU2015149979A (ru) 2010-03-30 2019-01-15 Октафарма Аг Способ очистки белка фактора роста
WO2011121020A1 (fr) 2010-03-30 2011-10-06 Octapharma Ag Procédé de purification de protéines dépendantes de la vitamine k telles que le facteur de coagulation ix
US9422329B2 (en) 2010-11-05 2016-08-23 Hoffmann-La Roche Inc. Optimized method for antibody capturing by mixed mode chromatography
CA2821711C (fr) 2010-12-15 2017-10-10 Baxter International Inc. Recuperation d'eluat par utilisation d'un gradient de conductivite
WO2013075740A1 (fr) 2011-11-23 2013-05-30 Sanofi Procédé de purification d'anticorps
US10188965B2 (en) 2012-12-05 2019-01-29 Csl Behring Gmbh Hydrophobic charge induction chromatographic depletion of a protein from a solution
US20140154233A1 (en) 2012-12-05 2014-06-05 Csl Limited Method of purifying therapeutic proteins
CN103396494B (zh) * 2013-04-27 2015-04-01 江苏省疾病预防控制中心 一种凝血因子ix的单克隆抗体
TWI631132B (zh) 2013-05-06 2018-08-01 賽諾菲公司 用於純化抗體之連續多步驟方法
TW201617360A (zh) * 2014-06-25 2016-05-16 桂格 史考特 布蘭克 用於純化蛋白質之方法及試劑
MX2017000862A (es) 2014-08-04 2017-05-01 Csl Ltd Formulacion de factor viii.
DK3265483T3 (da) 2015-03-06 2020-03-02 CSL Behring Lengnau AG Modificeret von Willebrand-faktor med forbedret halveringstid
GB201506117D0 (en) 2015-04-10 2015-05-27 Ge Healthcare Bio Sciences Ab Method for chromatography
GB201506113D0 (en) 2015-04-10 2015-05-27 Ge Healthcare Bio Sciences Ab Method for chromatography
EP3205665A1 (fr) * 2016-02-11 2017-08-16 Octapharma AG Procédé de séparation de facteur viii à partir de produits sanguins
GB201617240D0 (en) * 2016-10-11 2016-11-23 Profactor Pharma Ltd Purification process
CN111183151B (zh) 2017-06-23 2024-06-07 武田药品工业株式会社 因子viii亚种的纯化
KR20220029733A (ko) 2019-07-04 2022-03-08 체에스엘 베링 렝나우 아게 응고 인자 viii의 시험관내 안정성을 증가시키기 위한 절단된 폰 빌레브란트 인자 (vwf)
JP2023500953A (ja) 2019-11-11 2023-01-11 ツェー・エス・エル・ベーリング・レングナウ・アクチエンゲゼルシャフト 第viii因子に対する寛容を誘導するためのポリペプチド

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE9302006D0 (sv) * 1992-10-02 1993-06-11 Kabi Pharmacia Ab Protein formulation
SE9301581D0 (sv) * 1993-05-07 1993-05-07 Kabi Pharmacia Ab Protein formulation
SE503424C2 (sv) * 1994-11-14 1996-06-10 Pharmacia Ab Process för rening av rekombinant koagulationsfaktor VIII
IL157309A0 (en) * 2003-08-07 2004-02-19 Applied Research Systems Method for the purification of a non-immunoglobulin protein
EP1831242B1 (fr) * 2004-12-23 2012-09-26 Novo Nordisk Health Care AG Reduction du contenu en contaminants proteiques de compositions renfermant une proteine d'interet dependante de la vitamine k
EP1707634A1 (fr) 2005-03-29 2006-10-04 Octapharma AG Procédé pour l'isolation de protéines recombinantes
JP5619738B2 (ja) 2008-06-24 2014-11-05 オクタファルマ アクチェンゲゼルシャフト 凝固第viii因子の精製方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009007451A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11203747B2 (en) 2012-10-24 2021-12-21 Genzyme Corporation Elution of biomolecules from multi-modal resins using MES and MOPS as mobile phase modifiers

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BRPI0814220B8 (pt) 2021-05-25
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BRPI0814220A2 (pt) 2015-01-06
KR20100051620A (ko) 2010-05-17
AU2008274195B2 (en) 2013-09-05
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US20100204452A1 (en) 2010-08-12
CN101730707A (zh) 2010-06-09
CA2692814A1 (fr) 2009-01-15
KR101293504B1 (ko) 2013-08-07
CN101730707B (zh) 2014-12-17
JP5323072B2 (ja) 2013-10-23
US8399620B2 (en) 2013-03-19
BRPI0814220B1 (pt) 2019-09-17
ATE503763T1 (de) 2011-04-15
RU2493163C2 (ru) 2013-09-20
PL2167526T3 (pl) 2011-09-30
NL300663I2 (fr) 2016-09-22
EP2167526B1 (fr) 2011-03-30
WO2009007451A1 (fr) 2009-01-15
ES2364118T3 (es) 2011-08-25

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